Diagnostic antigen and a method of in vitro diagnosing an active infection caused by hepatitis C virus

ABSTRACT

A peptide of the formula (SEQ ID NO:1) Met Ser Thr Ash Pro Lys Pro Cys Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Cys Asp Val Lys Phe Pro Gly Gly, Gly wherein there is a disulfide bridge between the two cysteine residues, is described. Further, a diagnostic antigen in carrier-bound form comprising said peptide is disclosed. Said peptide may be used in a method of in vitro diagnosing an active infection caused by hepatitis C virus.

This application is the national stage of International Application no.PCT/SE94/01183, filed Dec. 8, 1994.

The present invention relates to a new peptide, to a new diagnosticantigen comprising said peptide and a method of in vitro diagnosing anactive infection caused by hepatitis C virus (HCV) which makes use of adiagnostic antigen according to the invention.

BACKGROUND

The hepatitis C virus is one of the most recently identified humanpathogenic viruses, first described in 1989 (Choo Q-L, et al., Science244:359-362 (1989); Kuo G, et al., Science 244:362-364 (1989)). HCV isone of the first of the viruses termed non-A, non-B viruses to beidentified. HCV seems to have been the major cause for posttransfusional hepatitis since the introduction of HBV screening at bloodbanks (Kuo, et al., ibid). The world wide spread of HCV has been shownto be similar to that of HBV. Several routs for parenteral infectionshave been shown, such as needlestick injuries, intravenous drug use, andthrough immune globulin preparations (Cariani E, et al., Lancet 337:850(1991); Chamot E, Aquired Immuno Deficiency Syndrome 6:430-431 (1992);Horst H. A., N Engl J Med 325:132-3 (1991)).

HCV has a size of 30-38 nm and is a member of the flaviviridae with aRNA coded genome of approximately 9.5 kilo bases. Based on the homology,the HCV genome has been proposed to code for two or three structuralproteins, core and envelope, and perhaps also a matrix protein(Takamizawa A, et al., Journal of Virology 65:1105-13 (1991)).

The structure of the HCV is yet unknown, it can only be assumed by theproposed homology with the other members of the flaviviridae. Proteinsrelated to HCV have only been produced as recombinant constructs, and nocomplete HCV virion has been observed. The proteins are estimated to betranslated at the following sizes: core 192 amino acids; possibly the 70carboxy terminal of these is the matrix protein, which is based on thehomology with members of the flaviviridae (Takamizawa, et al., ibid);the E1 192 amino acids, the E2-NS1 344 amino acids, the NS2 278 aminoacids, the NS3 609 amino acids, the NS4 398 amino acids, and the NS5 998amino acids.

The most variable regions of the HCV genome have been shown to residewithin the probable envelope genes, whereas the 5' end and the coreregions of the HCV genome seem to be highly conserved.

The main assays, so far, for studying the immunology of HCV has both inEurope and USA been the enzyme immuno assay (EIA) with the recombinantC-100 construct which covers parts of the NS4 protein (Kuo, et al.,ibid). More recently assays containing either long synthetic peptides orrecombinant peptides which cover both structural and non-structural HCVproducts, have been introduced (Hosein B, et al., Proc Natl Acad Sci USA88:3647-51 (1991); Mimms L, et al., Lancet 336:1590-1 (1990)). Theproblems with the early, first generation, assays were bothunsatisfactory sensitivity and specificity (Dawson G. J., et al.,Journal of Clinical Microbiology 29:551-556 (1991)), though the secondgeneration assays do seem to have improved the serology according tothese problems (Chaudhary R. K., et al., J Clin Lab Anal 7:164-7 (1993);Chaudhary R. K., et al., Journal of Clinical Microbiology 29:2329-2330(1991); Marcellin P, et al., Lancet 337:551-2 (1991)).

What is known about the immune response to HCV has mainly been obtainedby using these assays. Most persons infected by HCV develop antibodiesto one or more of the proteins, mainly the core and NS3/NS4 (Nasoff M.S., et al., Proc Natl Acad Sci USA 88:5462-6 (1991); Okamoto H, et al.,Virology 188:331-341 (1992); Okamoto H, et al., Japanese Journal ofExperimental Medicine 60:223-33 (1990); Sallberg M, et al., ImmunologyLetters 33:27-34 (1992); Sallberg M, et al., Journal of ClinicalMicrobiology 30:1989-1994 (1992)). Recombinant constructs covering theseregions are termed c22 (core; Chiba J, et al., Proc Natl Acad Sci88:4641-4645 (1991); Harada S, Journal of Virology 65:3015-21 (1991)),c33 (part of NS3), and C-100 (parts of NS3/NS4), and the C5-1-1 (a partof the C100-3 protein). The immune response to c22 and c33 haveshortened the serodiagnosis of HCV in acute infections (Mattson L, etal., Scandinavian Journal of Gastroenterology 26:1257-1262 (1992)). Nomarker has been found to correlate to chronic infection, and IgM todifferent proteins have so far not been fully successful to bedifferentiating between acute and chronic infection. Only the persistantdetection of HCV RNA by polymerase chain reaction (Garson J, et al.,Lancet 335:1419-1422 (1990); Okamoto H, et al., Japanese Journal ofExperimental Medicine 60:215-22 (1990); Weiner A. J., et al.,Proceedings of the National Academy of Sciences USA 89:3468-3472 (1992))has proven to be diagnostic for differentiating the acute from chroniccarriers. One observation making HCV serology difficult is thatseropositive individuals may loose their antibodies to HCV, orseronegative individuals may have HCV RNA. Immunity after HCV infectionseems to be rather weak.

DESCRIPTION OF THE INVENTION

The present invention provides a new peptide of the formula ##STR1##wherein there is a disulfide bridge between the two cysteine residues(SEQ ID NO: 1).

This synthetic peptide (HCV-15) has been chemically synthesized and theamino acid sequence thereof is similar to the N-terminal amino acids1-28 of the amino acid sequence disclosed by Takeuchi, K et al., inNucleic Acid Research 18:4626 (1990). However, the peptide of theinvention, HCV-15, has two cysteine residues at positions 8 and 20,respectively, instead of Gln, and has further a disulphide bridgebetween said two cysteine residues formed by a chemical oxidation step.

The invention is further directed to a diagnostic antigen incarrier-bound form comprising the peptide according to the invention,HCV-15. The carrier may be coupled to the peptide by any suitabletechnique known in the art. The term "carrier" should be interpretedbroadly, and it may be a surface, such as microtiter plate, glass orplastic beads, an amino acid residue, a peptide or a protein, such askeyhole limpet hemocyanine, bovine serum albumin, poly-L-lysine or acombination of such carriers as long as the carrier does not destroy theability of the peptide of the invention to bind to HCV antibodies.

The diagnostic antigen of the invention does not only detect antibodiesdirected to HCV in a sample of body fluid, such as blood, salive orurine, but makes it also possible to differentiate between past andongoing infection.

Thus, the invention is also directed to a method of in vitro diagnosingan active infection caused by hepatitis C virus which comprisessubjecting a sample of body fluid from an individual to be diagnosed toan immunoassay making use of a diagnostic antigen according to theinvention followed by evaluation of the level of reactivity obtained,low levels indicating past infection and high levels indicating activeinfection.

There are several known immunoassay techniques which can be used, suchas radioimmunoassay (RIA), enzyme immunoassay (EIA), blot assays, suchas Western blot, and agglutination assays, such as latex, particle andhemagglutinin. The detection methods are different in the differenttypes of techniques, making use of certain types of markers asappropriate, but all immunoassay techniques are based onantibody-antigen reactivity, i.e. the amount of such complexes formed inrelation to a standard or negative sample.

The diagnostic antigen according to the invention has been found todetect antibodies to the HCV core protein in more than 94% of personswith antibodies to the HCV (see Table 1). When compared to an anti-HCVcore reactivity detected by a commercial assay containing a recombinantHCV core protein, the sensitivity of the HCV-15 EIA assay was found tobe 89%-95% (see Tables 1 and 2).

When the method of in vitro diagnosing an active infection caused byhepatitis C virus of the invention was used, it was possible todiscriminate between active and past infection by determination of thelevel of reactivity. When testing 134 samples, out of which 129 werereactive in different commercial anti-HCV EIAs, 84 were found to bepositive for HCV RNA by PCR. Out of these 84 sera, 75 were reactive inthe HCV-15 assay. The reactivity to the HCV-15 peptide of the inventionwas found to be significantly related to the presence of HCV RNA, asdetermined by PCR (p<0.01; see Table 3).

Further, the mean level of reactivity in the HCV-15 assay was found tobe significantly higher in samples containing HCV RNA detected by thepolymerase chain reaction (see Table 4). Thus, a high level ofreactivity to the HCV-15 peptide is a sign of ongoing HCV infection.

Due to the high predictive value for the presence of HCV RNA when usingthe diagnostic antigen of the invention in an immunoassay, the method ofdiagnosing an active infection caused by HCV according to the inventionmay function as a rapid surrogate diagnosis for determining ongoinginfection (see Tables 3 and 4).

It should be mentioned that the diagnostic antigen of the invention,which is a single cyclized synthetic peptide, has a specificity which iscomparable to the presently available anti-HCV assays using multiplepeptides or multiple recombinant antigens (see Tables 3-5).

Synthesis of the peptide of the Invention

The peptide of the invention is first synthesized in linear form makinguse of a suitable method commonly known in the art, such as geneticengineering, or coupling of one amino acid residue to the next orcoupling of shorter sequenses in proper order, whereby peptide bonds areformed between residues, until the whole linear peptide is built-up,either in liquid medium or on a solid support (so-called solid phasesynthesis). Then the linear peptide is subjected to a chemical oxidationstep for ring-closure between the two cystein residues, whereby adisulfide bond is formed.

The solid phase technique was used for the synthesis of the peptide ofthe invention in accordance with the following referenses:

Merrifield, R. B. (1963) J. Am. Chem. Soc. 85:2149

Merrifield, R. B. (1964) Biochem. 3:1385

Konig, W. & Geiger, R. (1970) Chem. Ber. 103:788

Sheppard, R. C. (1973) In Nesvadba, H. (ed) Peptides 1971, NorthHolland, Amsterdam p. 111

Atherton, E., Gait, M. J., Sheppard, R. C. & Williams, B. J. (1979)Bioorg. Chem. 8:351

Sheppard, R. C. (1986) Science Tools 33:9-16

Atherton, E. & Sheppard, R. C. (1981) In Eberle, A., Geiger, R. &Wieland, T. (eds) Perspectives in Peptide Chemistry, Karger, Basel p.101.

In addition to established three-letter codes for the amino acids, thefollowing abbreviations are used:

    ______________________________________                                        Boc         tert-butoxycarbonyl                                               DIPCDI      diisopropyl carbodiimide                                          DMF         dimethylformamide                                                 EDT         ethanedithiol                                                     FAB-MS      fast atom bombardment mass spectrometry                           Fmoc        9-fluorenylmethoxycarbonyl                                        HOBt        1-hydroxybenzotriazole                                            OtBu        tert-butoxy                                                       Pmc         pentamethylchromansulfonyl                                        POE         polyoxyethylene                                                   tBu         tert-butyl                                                        TFA         trifluoroacetic acid                                              Trt         triphenylmethyl                                                   ______________________________________                                    

All the amino acids used during the synthesis were protected by aFmoc-group on the alpha-amino function. The following amino acids wereprotected in the side chain:

Thr(tBu), Ser(tBu), Asn(Trt), Cys(Trt), Lys(Boc), Asp(OtBu) andArg(Pmc).

The Amino acid derivatives were purchased from CalBiochem NovoBiochemGmbH, Badsoden, Germany.

The peptide of the invention having the formula SEQ ID NO: 1: ##STR2##wherein there is a disulfide bridge between the two cysteine residues,was synthesized in accordance with the solid phase technique undercontinuous flow on a Milligen 9050 Peptide Synthesizer (Millipore Corp.,Mass., USA) (Atherton, E., Sheppard, R. C. (1989) Solid Phase SynthesisA Practical Approach. Oxford, Oxford University Press.)

The resin used was of POE type with Rink-linker and a theoretical loadof 0.23 meq/g (Rapp Polymer, Tubingen, Germany). The amino acids wereactivated with DIPCDI/HOBt in DMF and the N(alpha)-Fmoc group wasremoved by 20% piperidine in DMF. The product of the synthesis was driedin vacuum overnight. The peptide was then cleaved from the resin bytreatment with TFA in the presence of EDT and phenol as scavengers(TFA:phenol:EDT 95:2.5:2.5). The TFA mixture and the peptide wereprecipitated by diethyl ether (100 ml) and filtrated. The precipitatewas washed on the filter with additional diethyl ether (3×30 ml) and thecleaved-off peptide was extracted with water (100 ml). The extract wasimmediately diluted to a volume of 1 dm³ with 20% acetic acid inmethanol and was treated with a 0.1 mole/1 solution of iodine inmethanol until a faint yellow colour persisted.

Dowex 1×8 ion exchanger in acetate form (3 g) (Biorad, Richmond, Calif.,USA) was then added, and the mixure was filtrated. The filtrate wassubjected to evaporation and the residue was lyophilized from water.

The product was isolated by liquid chromatography (reversed phase). Thestationary phase in the column consisted of Kromasil, 100 Å, C₈, 5 μ(EKA Nobel, Sweden; Hichrome Ltd, Reading, Berkshire, England), and themobile phase was acetonitrile/water containing 0.1% of TFA. The samplescollected from the coulmn were analyzed by an analytical HPLC (Varian5500, Sunnyvale, Calif., USA) which was equipped with an analyticalcolumn having the same stationary phase as the above described one.Those fractions containing pure substance (HPLC analysis) were pooledand the solvent was evaporated. The product was lyophilized from water.

The final HPLC analysis was made on ready product. Purity (HPLC): 99.9%

The structure was confirmed by FAB-MS. M+H!⁺ =3145 (M-Scan Ltd,Sunninghill, Ascot, Berkshire, England), and by amino acid analysis(AAA) (Malmo Allmanna Sjukhus, Institutionen for Klinisk Kemi, Malmo,Sweden).

    ______________________________________                                        AAA:                                                                          AA             obtained calculated                                            ______________________________________                                        Asp,Asn        3.93     4                                                     Arg            3.95     4                                                     Cystine        0.67     1                                                     Gly            3.16     3                                                     Lys            4.13     4                                                     Met            0.99     1                                                     Phe            1.01     1                                                     Pro            3.89     4                                                     Thr,Ser        3.84     4                                                     Val            1.00     1                                                     ______________________________________                                    

Detection of antibodies to HCV-15 by enzyme immunoassays (EIAs)

One-hundered μl of HCV-15 peptide was passively adsorbed overnight, atroom temperature, to polystyrene microtiter plates (Nunc Maxisorb 96FCertificate, Nunc, Roskilde, Denmark) at a concentration of 10 μgpeptide per mililiter of 0.05M sodium carbonate buffer, pH 9.6. Prior toaddition of 100 μl human serum diluted 1:100 in phosphate bufferedsaline (PBS) containing 1% bovine serum albumin (BSA), 2% goat serum(Sigma Chemicals, St. Louis, Mo.), and 0.05% Tween 20 (dilution buffer),the plates were washed four times with PBS containing 0.05% Tween 20.The diluted human serum samples were incubated on the microtiter platefor 45 minutes at ±37° C. After additional washing to remove unboundmaterial, 100 μl of alkaline phosphatase conjugated goat anti-human IgGdiluted (A-3150, Sigma Chemicals) 1:1500 in dilution buffer, was addedand incubated on the plate for 30 minutes at ±37° C. The plate was againwashed to remove excess material, and 100 μl of dinitrophenylenediamine(1 mg/ml) was added to each well, followed by incubation on the platefor 30 minutes at room temperature (20°-22° C.). The enzyme reaction wasthen terminated by addition of 100 μl 1M NaOH to each well, and theabsorbances were read at 405 nm using a double beam spectrophotometer.Absorbances exceeding the mean OD at 405 nm of at least 10 anti-HCVnegative human sera by more than three times their standard devationwere regarded as containing antibodies to the HCV-15 peptide.

                  TABLE 1                                                         ______________________________________                                        Relation between presence of antibodies to HCV in 2:nd generation             Abbott EIA (Abbott, Chicago, Ill.) and presence of antibodies to HCV-15       in 88 Italian sera (kindly provided by Dr. Armando Gabrielli, Ancona)                             No. of sera reac-                                                             tive in Abbott                                            No. of sera reac-   anti-HCV EIA                                              tive to HCV-15                                                                             +           -     total                                          ______________________________________                                        +            70           0    70                                             -             4          14    18                                             total        74          14    88                                             ______________________________________                                         p < 0.01, Fisher's exact test.                                                Note: Sensitivity: 95%                                                        Specificity: 100%                                                        

                  TABLE 2                                                         ______________________________________                                        Relation between antibody reactivity detected by the                          HCV-15 peptide EIA and Abbott Supplemental assay in                           96 human sera provided by SBL, Stockholm.                                                No. of sera reactive in                                                       Abbott Supplemental Assay                                          No. of sera reac-                                                                        Positive Indeterminat Negative                                     tive to HCV-15                                                                          S+/NS+   S+/NS-  S-/NS+ S/NS- Total                                 ______________________________________                                        +         41       9       1       1    52                                    -          1       4       4      35    44                                    Total     42       13      5      36    96                                    ______________________________________                                         Sensitivity 98% 69% 20% 97% (Specificity)                                     Abbreviations: S = bead coated with recombinant HCV core protein              NS = bead coated with recombinant HCV NS3 and NS4                             proteins                                                                      Total sensitivity 85%                                                    

                  TABLE 3                                                         ______________________________________                                        Relation between the presence of HCV RNA and mean                             sample to cut-off ratio (S/CO) in HCV peptide EIAs of                         positive reactions using human sera.                                                          No. sera                                                                              Mean                                                  Peptide                                                                              HCV      positive                                                                              S/CO        P-value                                   EIA    RNA      in EIA  ratio  SD   (Whitney-Mann)                            ______________________________________                                        HCV-15 +        75      6,17   2,29 0,0352                                           -        15      4,73   2,22                                           ______________________________________                                         Note: S/CO = the absorbance at 405 nm of the sample divided by                the mean of the negative sera plus three times their                          standard deviation.                                                      

                  TABLE 4                                                         ______________________________________                                        Relation between presence of HCV RNA by PCR and                               antibodies to HCV-15 in 134 Swedish sera (kindly                              provided by Dr. Anders Sonnerborg, SMCL, Stockholm).                          HCV     HCV-15                                                                RNA     +              -     Total                                            ______________________________________                                        +       75              9    84                                               -       15             35    50                                               Total   90             44    134                                              ______________________________________                                         p < 0.01, Fisher's exact test.                                                Note: Sensitivity: 89%                                                        Specificity: 80%                                                         

                  TABLE 5                                                         ______________________________________                                        Relation between Abbott Supplemental and Organon 2 in                         96 human sera obtained from SBL, Stockholm.                                             No. of sera reactive in                                             No. of sera reac-                                                                       Abbott Supplemental Assay                                           tive in Organon 2                                                                       Positive Indeterminat Negative                                      EIA       S+/NS+   S+/NS-  S-/NS+                                                                              S-/NS-                                                                              Total                                  ______________________________________                                        +         41       7       4      0    52                                     -          1       6       1     36    44                                     Total     42       13      5     36    96                                     ______________________________________                                         Sensitivity 98% 54% 80% 100% (Specificity)                                    Total sensitivity: 87%                                                   

                  TABLE 6                                                         ______________________________________                                        Relation between HCV-15 and Organon 2 in 96 human                             sera obtained from SBL, Stockholm.                                                           No. of sera reactive                                                          in Organon 2                                                   No. of sera reac-                                                                            anti-HCV EIA                                                   tive to HCV-15                                                                            +           -     total                                           ______________________________________                                        +           48           4    52                                              -            4          40    44                                              total       52          44    96                                              ______________________________________                                         Note: Sensitivity: 94%                                                        Specificity: 91%                                                         

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 8..20                                                           (D) OTHER INFORMATION: /note="DISULFIDE BRIDGE BETWEEN                        CYS IN POSITION 8 AND CYS IN POSITION 20"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetSerThrAsnProLysProCysArgLysThrLysArgAsnThrAsn                              151015                                                                        ArgArgProCysAspValLysPheProGlyGlyGly                                          2025                                                                          __________________________________________________________________________

We claim:
 1. A peptide of the formula ##STR3## wherein there is adisulfide bridge between the two cysteine residues (SEQ ID NO: 1).
 2. Adiagnostic antigen in carrier-bound form comprising a peptide accordingto claim
 1. 3. A method of in vitro diagnosing an active infectioncaused by hepatitis C virus which comprisessubjecting a sample of bodyfluid from an individual to be diagnosed to an immunoassay making use ofa diagnostic antigen according to claim 2 followed by evaluation of thelevel of reactivity obtained, high levels indicating active infection.